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UESAKA Toshihiro
Graduate School of Medicine / Faculty of Medical Sciences
Associate Professor

Researcher basic information

■ Research Areas
  • Life sciences / Neuroanatomy and physiology

Research activity information

■ Paper
  • Mukhamad Sunardi, Keisuke Ito, Yuya Sato, Toshihiro Uesaka, Mitsuhiro Iwasaki, Hideki Enomoto
    BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance in vivo has remained largely unclear. METHODS: We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. RESULTS: RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. CONCLUSIONS: This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations.
    Elsevier BV, Dec. 2022, Cellular and Molecular Gastroenterology and Hepatology, 15(6) (6), 1505 - 1524
    Link to Kobe Univ. Repository (Kernel)

  • Yuta Yoshioka, Yoshihisa Tachibana, Toshihiro Uesaka, Hiroyuki Hioki, Yuya Sato, Takumi Fukumoto, Hideki Enomoto
    Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.
    Jun. 2022, Biochemical and biophysical research communications, 608, 66 - 72
    Link to Kobe Univ. Repository (Kernel)

  • Toshihiro Uesaka, Mitsumasa Okamoto, Mayumi Nagashimada, Yoshihiro Tsuda, Miho Kihara, Hiroshi Kiyonari, Hideki Enomoto
    Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.
    Nov. 2021, Glia, 69(11) (11), 2575 - 2590

  • Taichi Nakatani, Mitsuhiro Iwasaki, Atsuhiro Yamamichi, Yuta Yoshioka, Toshihiro Uesaka, Yuko Bitoh, Kosaku Maeda, Takumi Fukumoto, Tatsuya Takemoto, Hideki Enomoto
    Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (RetM919T , RetN396K and RetY792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. RetM919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in RetM919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.
    May 2020, Development, growth & differentiation, 62(4) (4), 214 - 222
    Link to Kobe Univ. Repository (Kernel)

  • Mitsumasa Okamoto, Yuta Yoshioka, Kosaku Maeda, Yuko Bito, Takumi Fukumoto, Toshihiro Uesaka, Hideki Enomoto
    Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.
    May 2019, Genesis (New York, N.Y. : 2000), 57(5) (5), e23292
    Link to Kobe Univ. Repository (Kernel)

  • Heather M. Young, Lincon A. Stamp, Toshihiro Uesaka, Marlene M. Hao, Donald F. Newgreen, Hideki Enomoto
    Elsevier Inc., Mar. 2018, Physiology of the Gastrointestinal Tract: Sixth Edition, 1-2, 273 - 288

  • Toshihiro Uesaka, Mayumi Nagashimada, Hideki Enomoto
    Jul. 2015, JOURNAL OF NEUROSCIENCE, 35(27) (27), 9879 - 9888
    Link to Kobe Univ. Repository (Kernel)

  • Toshihiro Uesaka, Mayumi Nagashimada, Hideki Enomoto
    Oct. 2013, JOURNAL OF NEUROSCIENCE, 33(41) (41), 16372 - 16382

  • Chihiro Nishiyama, Toshihiro Uesaka, Takayuki Manabe, Yohei Yonekura, Takashi Nagasawa, Donald F. Newgreen, Heather M. Young, Hideki Enomoto
    Sep. 2012, NATURE NEUROSCIENCE, 15(9) (9), 1211 - U64

  • Mayumi Nagashimada, Hiroshi Ohta, Chong Li, Kazuki Nakao, Toshihiro Uesaka, Jean-Francois Brunet, Jeanne Amiel, Delphine Trochet, Teruhiko Wakayama, Hideki Enomoto
    Sep. 2012, JOURNAL OF CLINICAL INVESTIGATION, 122(9) (9), 3145 - 3158

  • Chihiro Nishiyama, Toshihiro Uesaka, Takayuki Manabe, Yohei Yonekura, Takashi Nagasawa, Donald F. Newgreen, Heather M. Young, Hideki Enomoto
    Sep. 2012, NATURE NEUROSCIENCE, 15(9) (9), 1211 - U64

  • Mayumi Nagashimada, Hiroshi Ohta, Chong Li, Kazuki Nakao, Toshihiro Uesaka, Jean-Francois Brunet, Jeanne Amiel, Delphine Trochet, Teruhiko Wakayama, Hideki Enomoto
    Sep. 2012, JOURNAL OF CLINICAL INVESTIGATION, 122(9) (9), 3145 - 3158

  • Toshihiro Uesaka, Hideki Enomoto
    Apr. 2010, Journal of Neuroscience, 30(15) (15), 5211 - 5218

  • Toshihiro Uesaka, Hideki Enomoto
    Apr. 2010, JOURNAL OF NEUROSCIENCE, 30(15) (15), 5211 - 5218

  • Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, Hideki Enomoto
    May 2008, JOURNAL OF CLINICAL INVESTIGATION, 118(5) (5), 1890 - 1898

  • Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, Hideki Enomoto
    May 2008, JOURNAL OF CLINICAL INVESTIGATION, 118(5) (5), 1890 - 1898

  • Toshihiro Uesaka, Sanjay Jain, Shigenobu Yonemura, Yasuo Uchiyama, Jeffrey Milbrandt, Hideki Enomoto
    Jun. 2007, DEVELOPMENT, 134(11) (11), 2171 - 2181

  • Toshihiro Uesaka, Sanjay Jain, Shigenobu Yonemura, Yasuo Uchiyama, Jeffrey Milbrandt, Hideki Enomoto
    Jun. 2007, DEVELOPMENT, 134(11) (11), 2171 - 2181

  • Bhupinder P. S. Vohra, Keiji Tsuji, Mayumi Nagashimada, Toshihiro Uesaka, Daniel Wind, Ming Fu, Jennifer Armon, Hideki Enomoto, Robert O. Heuckeroth
    Oct. 2006, DEVELOPMENTAL BIOLOGY, 298(1) (1), 259 - 271

  • T Uesaka, N Kageyama, H Watanabe
    Mar. 2004, JOURNAL OF MOLECULAR BIOLOGY, 337(3) (3), 647 - 660

  • A water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia suppresses azoxymethane-induction of colon cancers in male F344 rats
    HM Lu, E Kyo, T Uesaka, O Katoh, H Watanabe
    Mar. 2003, ONCOLOGY REPORTS, 10(2) (2), 375 - 379

  • T Uesaka, HM Lu, O Katoh, H Watanabe
    Oct. 2002, AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY, 283(4) (4), G840 - G847

  • Prevention of development of N,N '-dimethylhydrazine-induced colon tumors by a water-soluble extract from cultured medium of Ganoderma lacidum (Rei-shi) mycelia in male ICR mice
    HM Lu, E Kyo, T Uesaka, O Katoh, H Watanabe
    Feb. 2002, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 9(2) (2), 113 - 117

■ MISC
■ Books And Other Publications
  • Physiology of the Gastrointestinal Tract / Development of the Enteric Nervous System
    HeatherM.Young, LinconAStamp, Uesaka Toshihiro, MarleneM.Hao, DonalfE.Newgreen, HidekiEnomoto
    Academic Press, 2018

■ Lectures, oral presentations, etc.
  • 腸管神経系の形成不全後のシュワン細胞系譜からの腸管ニューロン産生
    UESAKA TOSHIHIRO
    第124回日本解剖学会総会・全国学術集会, Mar. 2019, 新潟

  • 腸管神経系の形成不全後のシュワン細胞系譜からの腸管ニューロン産生
    UESAKA TOSHIHIRO
    CSMIリトリート「若手道場」, Jan. 2019, 淡路

  • RET 活性化型変異 C618F は遺伝子量減少によりヒルシュスプルング病を誘導する
    ITO KEISUKE, 岡本 光正, UESAKA TOSHIHIRO, 前田 貢作, ENOMOTO HIDEKI
    CSMIリトリート「若手道場」, Jan. 2019, 淡路

  • Formation of enteric nervous system from Schwann cell precursors in mouse models of Hirschsprung disease
    UESAKA TOSHIHIRO
    第2回神戸理研・神戸大学合同シンポジウム, Jan. 2019, 神戸

  • 腸管神経系形成不全下におけるシュワン細胞系譜からのニューロン産生
    UESAKA TOSHIHIRO, ENOMOTO HIDEKI
    第94回日本解剖学会近畿支部学術集会, Nov. 2018, 神戸

  • RET 活性化型変異 C618F は遺伝子量減少によりヒルシュスプルング病を誘導する
    ITO KEISUKE, 岡本 光正, UESAKA TOSHIHIRO, 前田 貢作, ENOMOTO HIDEKI
    第94回日本解剖学会近畿支部学術集会, Nov. 2018, 神戸

  • 胎児期と生後における腸管神経系の形成
    UESAKA TOSHIHIRO
    第８回 Tokyo Vertebrate Morphology Meeting, Aug. 2018, 東京

  • Formation of enteric nervous system from Schwann cell precursors in mouse models of Hirschsprung disease
    Toshihiro Uesaka
    Development of the Enteric Nervous System:cells,signals,genes and therapy 5th International Symposium, Apr. 2018, Boston

  • Glial cell-based development and function of the enteric nervous system
    Enomoto Hideki, Uesaka Toshihiro
    第70回日本自律神経学会総会, Aug. 2017, 日本自律神経学会, 名古屋

  • Enteric neurogenesis from Schwann cell lineage in models of Hirschsprung disease
    Uesaka Toshihiro, Enomoto Hideki
    第40回日本神経科学大会, Jul. 2017, 日本神経科学学会, 千葉

  • Enteric neurogenesis from Schwann cell lineage in mouse models of Hirschsprung disease
    Uesaka Toshihiro, Mayumi Nagashimada, Enomoto Hideki
    BMB2015, Dec. 2015, 日本分子生物学会The Molecular Biology Society of Japan, 神戸

  • The impact of RET gain-of-function mutation on development of the enteric nervous system
    Mitsumasa Okamoto, Uesaka Toshihiro, Keisuke Itoh, Enomoto Hideki
    4th International Symposium on "Development of the enteric nervous system:Cells,Singnals,Genes and Therapy", Apr. 2015, ENS meeting scientific organizing committee:Robert Hofstra,Alan Burns(local organizer), Rotterdam, The Netherlands

  • Neurogenesis from Schwann cell lineage in mouse models of Hirschsprung disease
    Uesaka Toshihiro, Mayumi Nagashimada, Enomoto Hideki
    4th International Symposium on "Development of the enteric nervous system:Cells,Singnals,Genes and Therapy", Apr. 2015, ENS meeting scientific organizing committee:Robert Hofstra,Alan Burns(local organizer), Rotterdam, The Netherlands

  • Schwann cell precursor-like cells are a novel neuronal origin of the enteric nervous system
    Uesaka Toshihiro, Enomoto Hideki
    47th Annual Meeting for the Japanese Society of Development Biologists, May 2014, Japanese Society of Developmental Biologists, 名古屋

  • Schwann cell precursor-like cells from the mesentery are a cellular source of enteric neurons
    Uesaka Toshihiro, Enomoto Hideki
    Gordon Research Conferences Neural crest, Jul. 2013, Gordon Reserch Conference, Easton, USA, neural crest

  • Schwann cell precursor-like cells from the mesentery are a cellular origin of enteric neurons
    Uesaka Toshihiro, Enomoto Hideki
    Neuro2013, Jun. 2013, Japan Neuroscience Society, 京都, Cellular otigin

■ Affiliated Academic Society
  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

  • JAPANESE SOCIETY OF DEVELOPMENTAL BIOLOGISTS

  • THE JAPAN NEUROSCIENCE SOCIETY

■ Works
  • 黎明研究 「遺伝子導入による小腸放射線障害の軽減効果の検証」
    2003

■ Research Themes
  • シュワン細胞による腸管神経系の再形成の誘導
    上坂 敏弘
    日本学術振興会, 科学研究費助成事業, 基盤研究(C), 神戸大学, 01 Apr. 2021 - 31 Mar. 2024
    潜在的に腸管ニューロン産生能を有するシュワン細胞の特性を明らかにするために、シュワン細胞系譜の細胞を遺伝学的に標識するDesert hedgehog (Dhh)::CreERT2ドライバーを用いて、蛍光標識された細胞を腸管膜や腸管から回収を試みた。しかし、現時点では、一匹あたりから単離・回収される細胞数が少ないため、まだ遺伝子発現解析には至っていない。そこで最近報告されたマウスとヒトの単一細胞RNA-seqデータを用いてDhh陽性細胞の遺伝子発現プロファイルを解析した。その結果、マウスでは、Dhh遺伝子発現は腸管に入ると検出されないレベルに下がってしまうため、経時的な遺伝子発現変化などを追跡することが困難であることがわかった。そこで第一段階として、迷走神経堤由来の腸管グリア細胞とDhh陽性シュワン細胞系譜の細胞間で遺伝子発現を比較し、シュワン細胞系譜の細胞の有用な遺伝子マーカー候補のリストアップを進め、Dhh遺伝子に代わるマーカーの探索を進めている。また他の末梢神経系のシュワン細胞の単一細胞RNA-seqデータと比較して、シュワン細胞とは異なる遺伝子群についてもリストを作成している。一方で、ヒト胎児においては、Dhh陽性細胞が腸管においてしばらくの間検出されるため、経時的な追跡が可能であることがわかった。ヒトDhh陽性細胞群データを解析したところ、シュワン細胞様細胞、Nestin陽性細胞、bipotent（ニューロン・グリア）様の三つのクラスターに分類できた。さらに迷走神経堤由来の細胞と比較し、ニューロン産生能の活性化に関わる分子群の探索に活用していく。

  • 上坂 敏弘
    科学研究費補助金／新学術領域研究, Apr. 2017 - Mar. 2019, Principal investigator
    Competitive research funding

  • 上坂 敏弘
    学術研究助成基金助成金／基盤研究(C), Apr. 2016 - Mar. 2019, Principal investigator
    Competitive research funding

  • 神経芽腫に対する新規治療剤の探索
    榎本 秀樹
    国立研究開発法人日本医療研究開発機構, 創薬支援推進事業・創薬総合支援事業, 2017
    Competitive research funding

  • Vascular tissues that supports migration of enteric neural progenitors
    ENOMOTO HIDEKI, UESAKA Toshihiro, ITO Keisuke
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), 01 Apr. 2010 - 31 Mar. 2015
    In this study we conducted cellular and molecular analyses on neuro-vascular interactions in the development of the enteric nervous system (ENS). By performing live-cell imaging of enteric neural progenitors and vasculature in mice, we examined cell behavior in developing ENS. We have discovered that a subset of enteric neural progenitors crosses the mesentery when the small and large intestines are juxtaposed to each other during development. Trans-mesenteric enteric neural progenitors are the principal source of the ENS in the colon, and impaired trans-mesenteric migration delays colonization of the colon by enteric neural progenitors. Moreover, trans-mesenteric enteric progenitors are reduced in a mouse model of Hirschsprung disease (HSCR: congenital absence of the ENS in the distal gut), suggesting a potential involvement of impaired trans-mesenteric migration in HSCR pathogenesis. We have also discovered that GDNF is one of the molecules that regulate trans-mesenteric migration.

  • 上坂 敏弘
    文部科学省, 科学研究費補助金(基盤研究(C)), 2013 - 2015, Principal investigator
    Competitive research funding

  • Toshihiro UESAKA
    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Grant-in-Aid for Scientific Research (C), The Institute of Physical and Chemical Research, 2008 - 2010, Principal investigator
    RET tyrosine kinase is required for the development of the enteric nervous system (ENS). Hypomorphic RET alleles cause intestinal aganglionosis [Hirschsprung disease (HSCR)]. We have shown that elevated expression of Bcl-xL inhibits ENS precursor death in both Ret-null and hypomorphic states. Moreover, the prevention of cell death allows morphologically and functionally normal ENS formation in Ret-hypomorphic mice. These results indicate that ENS precursor death is a principal cause of intestinal aganglionosis in a Ret hypomorphic state, and suggest that the inhibition of cell death is a ro...
    Competitive research funding

  • 上坂 敏弘
    文部科学省, 科学研究費補助金(若手研究(B)), 2006 - 2007, Principal investigator
    神経堤細胞が腸管全体に移行し、神経分化をすでに開始しているマウス胚15.5日目にGDNF受容体を条件的ノックアウトしたところ、結腸遠位部以降において36時間以内に神経網が消失することを見いだした。つまり、生体内でGDNFシグナルが腸神経細胞の生存に必須であることを初めて示したことになる。ヒルシュスプルング病における腸管壁内神経節細胞の先天的欠損は神経堤細胞の移行異常によるものと従来考えられてきたが、我々の研究により、GDNF/RETシグナルの減少による神経細胞死の可能性が新たに出てきた。そこで、Ret遺伝子変異によるヒルシュスプルング病モデルマウスを作製し、細胞死の有無を検証した。我々は、マウスにおいてRETチロシンキナーゼの発現を通常の約30%に下げることで、従来のRetノックアウトマウスと異なり、腎臓やモーターニューロンの発生異常は認められず、結腸遠位部以降の腸神経の欠損が認められ、またこの異常の発症率がオスに高い傾向がみられる
    Competitive research funding

  • 腸管神経の発生に関する研究
    2004
    Competitive research funding

  • Study on the development of the enteric nervous system
    2004
    Competitive research funding

  • 上坂 敏弘
    文部科学省, 科学研究費補助金(若手研究(B)), 2002 - 2003, Principal investigator
    主に腸で発現する転写調節因子Cdx2に着目し、Cdx2の標的遺伝子を明らかにしていくことによって、腸上皮細胞の細胞増殖、分化制御、さらには細胞死制御機構の解明と分化マーカーの同定を目指した。Cdx2の発現制御が可能なラット胎児由来未分化腸上皮細胞株IEC-6 Tet-Off/Cdx2を用いて、Cdx2の発現誘導に伴い転写促進もしくは転写抑制される遺伝子をオリゴヌクレオチド・マイクロアレイ(CodeLink UniSet Rat I Bioarray, Amersham)を用いて9,906種の遺伝子について調べた。現在23種類の遺伝子がCdx2によって発現上昇してくることをRT-PCRによって確認している。これらの内、ゲノム情報がわかっているものに関しては、プロモーター領域と予想される上流域においてCdx2結合部位配列(TTTAC/T)の検索を行った。この結果をもとに、ルシフェラーゼを用いたプロモーターアッセイ、ゲルシフトアッセイ、クロマチン沈降法を行い、Cdx2によって直接転写調節されることを確認した。これにより、転写調節因子E2F3、17
    Competitive research funding

  • アポトーシス抑制遺伝子MCL1の転写調節機構に関する研究
    加藤 修, 上坂 敏弘
    日本学術振興会, 科学研究費助成事業, 特定領域研究(C), 広島大学, 2000 - 2000
    放射線や抗がん剤によるがん細胞のアポトーシスを血管内皮細胞増殖因子(VEGF)のような細胞増殖因子が抑制すること、その分子機序のひとつとしてアポトーシス抑制作用を持つmcl1遺伝子の発現誘導が関与していることを我々はすでに明らかにしている(Katoh,o.et al,Cancer Res,55:5687-5692,1995;Katoh,o.et al,Cancer Res,58:5565-5569,1998)。また、放射線や抗がん剤に曝露されることによりmcl1遺伝子が発現誘導されることも知られている。mcl1遺伝子はbcl2ファミリーに属し、その遺伝子産物はアポトーシス抑制作用を持っている。がん細胞において放射線や抗がん剤曝露あるいは細胞増殖因子刺激によりmcl1遺伝子は、そのmRNA発現が誘導され、がん細胞の細胞死の抑制すなわち生存維持に働き、最終的にはがん治療に対する耐性獲得に関与している。上記した種々のストレスや刺激に対するmcl1遺伝子の転写調節機構を明らかにすることを目的として本研究を行った。マウスmcl1遺伝子5'側上流域の約1.2kbのDNAをクローニングし、塩基配列を決定した。さらに放射線曝露や細胞増殖因子刺激によるmcl1遺伝子の転写調節領域を明らかにするために、レポーターアッセイをおこなった。その結果、マウス白血病細胞株32Dあるいは血管内皮細胞株PmTc-1いずれを用いても放射線曝露刺激においては翻訳開始点から上流-256から-406の領域が活性上昇に重要であることが明らかになった。さらに血管内皮細胞増殖因子(VEGF)刺激においても同じ領域が活性上昇に重要であることを確認した。mcl1遺伝子の転写調節にはこの領域に結合する転写因子が関与している可能性が示唆された。

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